Genetic Engineering Lecture Notes PDF

GENETIC ENGINEERING IN T ROD UC T ION & R EST RI C TI ON ENZYMES OBJECTIVES You must be able to: Define: Restriction Enzymes (RE’s); Palindromes Restriction Mapping Outline how RE’s work Genetic Engineering: Recombinant DNA Technology Recombinant DNA technology : The manipulation a...

GENETIC ENGINEERING IN T ROD UC T ION & R EST RI C TI ON ENZYMES OBJECTIVES You must be able to: Define: Restriction Enzymes (RE’s); Palindromes Restriction Mapping Outline how RE’s work Genetic Engineering: Recombinant DNA Technology Recombinant DNA technology : The manipulation and combination of DNA molecules from different sources 3 MAJOR STEPS Restriction Enzyme use: Recombinant DNA technology requires the use of molecular scissors called restriction enzymes, which cut DNA at specific sequences. Gel electrophoresis: Separation of macromolecules DNA cloning: Two methods Use of plasmids and PCR What are Restriction Enzymes? Restriction enzymes are DNA cutting enzymes found in bacteria as a defense against invasion from foreign DNA (harvested from them). Also called Restriction Endonucleases A restriction enzyme recognizes and cuts DNA only a particular sequence of nucleotides. Bacteria prevent their own DNA being cut by modifying their nucleotides via DNA methylation  Palindromes While recognition sequences vary widely, with lengths between 4 and 8 nucleotides, many of them are palindromic. That is, the sequence on one strand reads the same in the reverse direction on the complementary strand. Nomenclature This follows a general pattern: 1. The first letter of the name of the genus in which a given enzyme is discovered, is written in capital letters. 2. This is followed by the first two letters of the species name of the organism. All three letters are written in italics. Eg Eco from Escherichia coli; Hin from Haemophilus influenza 3. The strain or identification type is depicted as a subscript Named Example EcoRI E genus Escherichia co species coli R strain RY13 I first endonuclease identified Restriction Mapping This is the process of obtaining structural information on a piece of DNA by the use of restriction enzymes. A restriction map is a description of restriction endonuclease cleavage sites within a piece of DNA. Restriction mapping involves digesting DNA with a series of restriction enzymes and then separating the DNA fragments by agarose gel electrophoresis. The distance between enzyme sites can be determined by the size of the DNA fragments Restriction Mapping Restriction mapping Multiple RE’s used Each RE cuts at a different location Fragments are separated by gel electrophoresis and move through the gel according to size Each RE fragment creates a signature band Uses of Restriction Enzymes Creating Genome Libraries – Recombinant DNA Technology Cloning DNA molecules Studying nucleotide sequences GENETIC ENGINEERING RECOMBINANT DNA TECHNOLOGY What is recombinant DNA technology? Recombinant DNA technology: joining together of DNA molecules from two different species that are inserted into a host organism to produce new genetic combinations that are of value to science, medicine industry and agriculture. Gel Electrophoresis: Isolation of DNA fragments Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge. It is used in molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge. Gel electrophoresis apparatus Gel electrophoresis apparatus – an agarose gel is placed in this buffer-filled box and an electrical field is applied via the power supply to the rear. The negative terminal is at the far end (black wire), so DNA migrates toward the cathode (red wire). INTERPRETATION OF RESULTS PCR: Requirements DNA Template DNA Polymerase Primers Nucleotides Reverse Transcription- PCR How PCR Works: Genetic Engineering: Application Medicine: Production of pharmaceuticals & Gene Therapy Agriculture: Plants and Animal Husbandry Creating Pharmaceutical Products: eg Human Insulin Gene Therapy Although gene therapy is a promising treatment option for a number of diseases (including inherited disorders, some types of cancer, and certain viral infections), the technique remains risky and is still under study to make sure that it will be safe and effective. Gene therapy is currently only being tested for the treatment of diseases that have no other cures. Plant Genetic Engineering A: Crop Improvement B: Genetically engineered traits: Herbicide, insect & virus resistance; altered oil content; delayed fruit ripening; pollen control C: Weather-resistant crops D: Genetically engineered food: Soybeans; corn; cotton  Animal Husbandry Transgenic animals model advancements in DNA technology in their development. The mechanism for creating one can be described in three steps: Healthy egg cells are removed from a female of the host animal and fertilized in the laboratory. The desired gene from another species is identified, isolated, and cloned. The cloned genes are injected directly into the eggs, which are then surgically implanted in the host female, where the embryo undergoes a normal development process.